EP2647388A1 - Anticorps dirigés contre l'ERBB3 et leurs utilisations - Google Patents

Anticorps dirigés contre l'ERBB3 et leurs utilisations Download PDF

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EP2647388A1
EP2647388A1 EP13174963.2A EP13174963A EP2647388A1 EP 2647388 A1 EP2647388 A1 EP 2647388A1 EP 13174963 A EP13174963 A EP 13174963A EP 2647388 A1 EP2647388 A1 EP 2647388A1
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Prior art keywords
antibody
erbb3
seq
antigen binding
variable region
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Birgit Schoeberl
Ulrik Nielsen
Michael Feldhaus
David Buckler
Arumugam Muruganandam
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Merrimack Pharmaceuticals Inc
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Merrimack Pharmaceuticals Inc
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
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    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the ErbB/HER subfamily of polypeptide growth factor receptors include the epidermal growth factor (EGF) receptor (EGFR, ErbB 1/HER1), the neu oncogene product (ErbB2/HER2), and the more recently identified ErbB3/HER3 and ErbB4/HER4 receptor proteins (see, e.g., Hynes et. al. (1994) Biochim. Biophys. Acta Rev. Cancer 1198, 165-184 ).
  • Each of these receptors is predicted to consist of an extracellular ligand-binding domain, a membrane-spanning domain, a cytosolic protein tyrosine kinase (PTK) domain and a C-terminal phosphorylation domain (see, e.g., Kim et al., (1998) Biochem. J. 334, 189-195 ).
  • PTK cytosolic protein tyrosine kinase
  • ErbB3 is constitutively phosphorylated on tyrosine residues in a subset of human breast cancer cell lines overexpressing this protein (see, e.g ., Kraus et al. (1993) Proc. Natl. Acad Sci. USA. 90, 2900-2904 ; and Kim et al. Supra; see, also, Schaefer et al. (2006) Neoplasia 8(7):613-22 and Schaefer et al. Cancer Res (2004) 64(10):3395-405 ).
  • ErbB3 remains largely unappreciated as a target for clinical intervention.
  • Current immunotherapies primarily focus on inhibiting the action of ErbB2 and, in particular, heterodimerization of ErbB2/ErbB3 complexes (see, e.g ., Sliwkowski et al. (1994) J. Biol. Chem. 269(20):14661-14665 (1994 )). Accordingly, it is an object of the present invention to provide improved immunotherapies that effectively inhibit ErbB3 signaling, and can be used to treat and diagnose a variety of cancers.
  • the present invention provides a novel class of monoclonal antibodies which binds to the ErbB3 receptor and inhibits various ErbB3 functions.
  • the antibodies described herein are capable of binding to ErbB3 and inhibiting EGF-like ligand mediated phosphorylation of the receptor.
  • EGF-like ligands include EGF, TGF- ⁇ , betacellulin, heparin-binding epidermal growth factor, biregulin and amphiregulin, which bind to EGFR and induce dimerization of EGFR with ErbB3. This dimerization, in turn, causes phosphorylation of ErbB3, and activates signaling through the receptor.
  • Monoclonal antibodies of the present invention are useful for treating and diagnosing a variety of cancers associated with ErbB3-mediated cellular signaling. Accordingly, in one embodiment, the present invention provides monoclonal antibodies (and antigen binding portions thereof) which bind to ErbB3 and inhibit EGF-like ligand mediated phosphorylation of ErbB3.
  • the antibodies are further characterized by one or more of the following properties: (i) inhibition of ErbB3 ligand-mediated signaling, including signaling mediated by binding of ErbB3 ligands, such as heregulin, epiregulin, epigen and BIR, to ErbB3; (ii) inhibition of proliferation of cells expressing ErbB3; (iii) the ability to decrease levels of ErbB3 on cell surfaces ( e.g ., by inducing internalization of ErbB3); (iv) inhibition of VEGF secretion of cells expressing ErbB3; (v) inhibition of the migration of cells expressing ErbB3; (vi) inhibition of spheroid growth of cells expressing ErbB3; and/or (vii) binding to an epitope located on domain I (residues 20-209) of ErbB3, for example, an epitope involving or spanning residues 20-202 of the amino acid sequence of ErbB3.
  • ErbB3 ligand-mediated signaling
  • Particular monoclonal antibodies and antigen binding portions thereof of the present invention exhibit a K D of 50 nM or less, as measured by a surface plasmon resonance assay or a cell binding assay
  • particular monoclonal antibodies and antigen binding portions thereof of the present invention include a heavy chain variable region comprising an amino acid sequence which is at least 80% (e.g ., 85%, 90%, 95%, 96%, 97%, 98% or 99%) identical to the heavy chain variable region amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:35, or SEQ ID NO: 37.
  • antibodies and antigen binding portions thereof of the present invention include a light chain variable region comprising an amino acid sequence which is at least 80% (e.g ., 85%, 90%, 95%, 96%, 97%, 98% or 99%) identical to the light chain variable region amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:36, or SEQ ID NO:38.
  • the antibodies may also include both of the aforementioned heavy chain and light chain variable regions.
  • variable heavy and light chain regions of the antibodies or antigen binding portions thereof typically include one or more complementarity determining regions (CDRs). These include one or more CDR1, CDR2, and CDR3 regions. Accordingly, other particular antibodies and antigen binding portions thereof of the present invention include one or more CDR sequences selected from a heavy chain variable region CDR1 comprising SEQ ID NO:7; a heavy chain variable region CDR2 comprising SEQ ID NO:8; a heavy chain variable region CDR3 comprising SEQ ID NO:9; a light chain variable region CDR1 comprising SEQ ID NO:10; a light chain variable region CDR2 comprising SEQ ID NO:11; a light chain variable region CDR3 comprising SEQ ID NO:12; and combinations thereof.
  • CDRs complementarity determining regions
  • Still other particular antibodies and antigen binding portions thereof of the present invention include one or more CDR sequences selected from a heavy chain variable region CDR1 comprising SEQ ID NO:13; a heavy chain variable region CDR2 comprising SEQ ID NO:14; a heavy chain variable region CDR3 comprising SEQ ID NO:15; a light chain variable region CDR1 comprising SEQ ID NO:16; a light chain variable region CDR2 comprising SEQ ID NO:17; a light chain variable region CDR3 comprising SEQ ID NO:18; and combinations thereof.
  • Still other particular antibodies and antigen binding portions thereof of the present invention include; or one or more CDR sequences selected from a heavy chain variable region CDR1 comprising SEQ ID NO:19; a heavy chain variable region CDR2 comprising SEQ ID NO:20; a heavy chain variable region CDR3 comprising SEQ ID NO:21; a light chain variable region CDR1 comprising SEQ ID NO:22; a light chain variable region CDR2 comprising SEQ ID NO:23; a light chain variable region CDR3 comprising SEQ ID NO:24; and combinations thereof.
  • Still other particular antibodies and antigen binding portions thereof of the present invention include; or one or more CDR sequences selected from a heavy chain variable region CDR1 comprising SEQ ID NO:39; a heavy chain variable region CDR2 comprising SEQ ID NO:40; a heavy chain variable region CDR3 comprising SEQ ID NO:41; a light chain variable region CDR1 comprising SEQ ID NO:42; a light chain variable region CDR2 comprising SEQ ID NO:43; a light chain variable region CDR3 comprising SEQ ID NO:44; and combinations thereof.
  • Still other particular antibodies and antigen binding portions thereof of the present invention include; or one or more CDR sequences selected from a heavy chain variable region CDR1 comprising SEQ ID NO:45; a heavy chain variable region CDR2 comprising SEQ ID NO:46; a heavy chain variable region CDR3 comprising SEQ ID NO:47; a light chain variable region CDR1 comprising SEQ ID NO:48; a light chain variable region CDR2 comprising SEQ ID NO:49; a light chain variable region CDR3 comprising SEQ ID NO:50; and combinations thereof.
  • the antibodies and antigen binding portions thereof may also comprise one or more CDRs which are at least 80% (e.g ., 85%, 90%, 95%, 96%, 97%, 98% or 99%) identical to any of the aforementioned CDRs, or combinations of CDRs.
  • the antibodies and antibody portions thereof are fully human (i.e ., contains human CDR and framework sequences).
  • Particular human antibodies of the present invention include those having a heavy chain variable region that is from a human VH3 germ line gene, and/or a light chain variable region from human VL2 germ line gene.
  • Also encompassed by the present invention are monoclonal antibodies and portions thereof that bind to the same or overlapping epitopes bound by any of the antibodies or portions thereof described herein (e.g., an epitope located on domain I of ErbB3, such as an epitope involving or spanning, residues 20-202 of the amino acid sequence of ErbB3).
  • Antibodies which have the same activity as the antibodies described herein, e.g., antibodies having the same sequence as Ab #6, are also encompassed by the present invention.
  • Antibodies of the present invention include all known forms of antibodies and other protein scaffolds with antibody-like properties.
  • the antibody can be a human antibody, a humanized antibody, a bispecific antibody, a chimeric antibody or a protein scaffold with antibody-like properties, such as fibronectin or Ankyrin repeats.
  • the antibody also can be a Fab, Fab'2, ScFv, SMIP, affibody, nanobody, or a domain antibody.
  • the antibody also can have any of the following isotypes: IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD, and IgE.
  • the present invention further provides compositions comprising combinations of antibodies or antigen binding portions described herein, formulated with an acceptable carrier and/or adjuvant.
  • the composition comprises two or more antibodies that bind different epitopes on ErbB3 or antibodies described herein combined with anti-cancer antibodies which do not bind ErbB3.
  • the present invention provides isolated nucleic acids encoding the antibodies and antigen binding portions thereof described herein.
  • the nucleic acid encodes a heavy chain variable region comprising a nucleotide sequence which is at least 80% ( e.g ., 85%, 90%, 95%, 96%, 97%, 98% or 99%) identical to, or which hybridizes under high stringency conditions to, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:35, or SEQ ID NO:37; or a light chain variable region comprising a nucleotide sequence which is at least 80% ( e.g ., 85%, 90%, 95%, 96%, 97%, 98% or 99%) identical to, or which hybridizes under high stringency conditions to, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:36, or SEQ ID NO:38; or combinations of such heavy and light variable regions.
  • the present invention further provides transgenic non-human mammals, hybridomas, and transgenic plants that express and/or produce the antibodies and antigen binding portions described herein.
  • kits comprising one or more isolated monoclonal antibodies or antigen binding portions thereof described herein and, optionally, instructions for use in treating or diagnosing a disease associated with ErbB3 dependent signaling, such as cancers.
  • Antibodies and antigen binding portions thereof of the present invention can be used in a broad variety of therapeutic and diagnostic applications, particularly oncological applications. Accordingly, in another aspect, the invention provides method for inhibiting EGF-like ligand mediated phosphorylation of ErbB3 in a subject by administering one or more antibodies or antigen binding portions thereof described herein in an amount sufficient to inhibit EGF-like mediated phosphorylation of ErbB3.
  • the invention further provides methods for treating a variety of cancers in a subject, including, but not limited to, melanoma, breast cancer, ovarian cancer, renal carcinoma, gastrointestinal/colon cancer, lung cancer, clear cell sarcoma, and prostate cancer, by administering one or more antibodies or antigen binding portions thereof described herein in an amount sufficient to treat the cancer.
  • the antibodies or antigen binding portions thereof can be administered alone or in combination with other therapeutic agents, such as anti-cancer agents, e.g ., other antibodies, chemotherapeutic agents and/or radiation.
  • the invention provides methods for diagnosing and prognosing diseases (e.g ., cancers) associated with ErbB3. In one embodiment, this is achieved by contacting antibodies or antigen binding portions of the invention (e.g ., ex vivo or in vivo) with cells from the subject, and measuring the level of binding to ErbB3 on the cells, wherein abnormally high levels of binding to ErbB3 indicate that the subject has a cancer associated with ErbB3.
  • diseases e.g ., cancers
  • this is achieved by contacting antibodies or antigen binding portions of the invention (e.g ., ex vivo or in vivo) with cells from the subject, and measuring the level of binding to ErbB3 on the cells, wherein abnormally high levels of binding to ErbB3 indicate that the subject has a cancer associated with ErbB3.
  • ErbB3 refers human ErbB3 protein, as described in U.S. Pat. No. 5,480,968 and Plowman et al., Proc. Natl. Acad. Sci. USA, 87:4905-4909 (1990 ); see, also, Kani et al., Biochemistry 44:15842-857 (2005 ), Cho and Leahy, Science 297:1330-1333 (2002 )).
  • EGF-like ligand refers to ligands of epidermal growth factor receptor (EGFR), including epidermal growth factor (EGF) and closely related proteins, such as transforming growth factor- ⁇ (TGF- ⁇ ), betacellulin (BTC), heparin-binding epidermal growth factor (HB-EGF), biregulin (BIR) and amphiregulin (AR), which bind to EGFR on the surface of cells and stimulate the receptor's intrinsic protein-tyrosine kinase activity.
  • EGF- ⁇ transforming growth factor- ⁇
  • BTC betacellulin
  • HB-EGF heparin-binding epidermal growth factor
  • BIR biregulin
  • AR amphiregulin
  • EGF-like ligands induce formation of EGFR (also referred to as ErbB1) and ErbB3 protein complex (see e.g., Kim et al., (1998) Biochem J., 334:189-195 ), which results in phosphorylation of tyrosine residues in the complex.
  • EGFR also referred to as ErbB1
  • ErbB3 protein complex see e.g., Kim et al., (1998) Biochem J., 334:189-195 , which results in phosphorylation of tyrosine residues in the complex.
  • the antibodies and antigen binding portions thereof of the present invention inhibit EGF-like ligand mediated phosphorylation of ErbB3 and, in certain embodiments, exhibit one or more of the following additional properties: (i) inhibition of one or more of heregulin, epiregulin, epigen and biregulin (BIR)-mediated signaling through ErbB3; (ii) inhibition of proliferation of cells expressing ErbB3; (iii) the ability to decrease levels of ErbB3 on cell surfaces; (iv) inhibition of VEGF secretion of cells expressing ErbB3; (v) inhibition of the migration of cells expressing ErbB3; (vi) inhibition of spheroid growth of cells expressing ErbB3; and/or (vii) binding to an epitope located on domain I of ErbB3, e.g., an epitope which involves or spans residues 20-202 of the amino acid sequence of ErbB3.
  • BIR biregulin
  • inhibitor refers to any statistically significant decrease in biological activity, including full blocking of the activity.
  • “inhibition” can refer to a decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% in biological activity.
  • the phrase "inhibition of EGF-like ligand mediated phosphorylation of ErbB3,” as used herein, refers to the ability of an antibody or antigen binding portion to statistically significantly decrease the phosphorylation of ErbB3 induced by an EGF-like ligand, relative to the phosphorylation in an untreated (control) cell.
  • the cell which expresses ErbB3 can be a naturally occurring cell or cell line or can be recombinantly produced by introducing nucleic acid encoding ErbB3 into a host cell.
  • the antibody or antigen binding portion thereof inhibits EGF-like ligand mediated phosphorylation of ErbB3 by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or 100%, as determined, for example, by Western blotting followed by probing with an anti-phosphotyrosine antibody as described in Kim et al., (1998) Biochem J., 334:189-195 and the Examples infra.
  • the phrase "inhibition of heregulin, epiregulin, epigen or biregulin-mediated signaling through ErbB3,” as used herein, refers to the ability of an antibody or an antigen-binding portion thereof to statistically significantly decrease signaling mediated by an ErbB3 ligand ( e.g ., heregulin, epiregullin, epigen and biregulin) through ErbB3, relative to the signaling in the absence of the antibody (control).
  • an ErbB3 ligand e.g ., heregulin, epiregullin, epigen and biregulin
  • ErbB3-ligands are also referred to herein as "heregulin-like ligands.” This means that, in the presence of the antibody or antigen binding portion thereof, a signal mediated in a cell expressing ErbB3 by one or more of heregulin, epiregulin, epigen and biregulin, relative to a control (no antibody), is statistically significantly decreased.
  • An ErbB3-ligand mediated signal can be measured by assaying for the level or activity of an ErbB3 substrate, and/or a protein which is present in a cellular cascade involving ErbB3.
  • the antibody or antigen binding portion thereof decreases the level or activity of an ErbB3 substrate and/or that of a protein in a cellular cascade involving ErbB3, by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or 100% relative to the level or activity in the absence of such antibody or antigen binding portion thereof (control).
  • ErbB3-ligand mediated signaling can be measured using art recognized techniques which measure the level or activity of a substrate of ErbB3 (e.g ., SHC or PI3K) or a protein in a cellular cascade involving ErbB3 (e.g ., AKT) using kinase assays for such proteins (see, e.g. , Horst et al. supra, Sudo et al. (2000) Methods Enzymol, 322:388-92 ; and Morgan et al. (1990) Eur. J. Biochem., 191:761-767 ).
  • a substrate of ErbB3 e.g ., SHC or PI3K
  • a protein in a cellular cascade involving ErbB3 e.g ., AKT
  • the antibody or antigen binding portion thereof inhibits ErbB3-ligand (e.g ., heregulin, epiregulin, epigen or biregulin) mediated signaling through ErbB3 by inhibiting the binding of the ErbB3-ligand (e.g ., one or more of heregulin, epiregulin, epigen or biregulin) to ErbB3.
  • ErbB3-ligand e.g ., one or more of heregulin, epiregulin, epigen or biregulin
  • Some ligands e.g ., biregulin or BIR
  • inhibitors refers to the ability of an antibody or an antigen-binding portion thereof to statistically significantly decrease the binding of an ErbB3 ligand (e.g ., one or more of heregulin, epiregulin, epigen or biregulin) to ErbB3, relative to the binding in the absence of the antibody (control).
  • an ErbB3 ligand e.g ., one or more of heregulin, epiregulin, epigen or biregulin
  • the amount of the ErbB3-ligand e.g ., heregulin, epiregulin, epigen or biregulin
  • the amount of an ErbB3 ligand which binds ErbB3 may be decreased in the presence of an antibody or antigen binding portion thereof of the invention by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or 100% relative to the amount in the absence of the antibody or antigen binding portion thereof (control).
  • a decrease in ErbB3-ligand binding can be measured using art recognized techniques which measure the level of binding of labeled ErbB3-ligand (e.g ., radiolabelled heregulin, epiregulin, epigen or biregulin) to cells expressing ErbB3 in the presence or absence (control) of the antibody or antigen binding portion thereof.
  • labeled ErbB3-ligand e.g ., radiolabelled heregulin, epiregulin, epigen or biregulin
  • the phrase "inhibition of proliferation of a cell expressing ErbB3,” as used herein, refers to the ability of an antibody or an antigen-binding portion thereof to statistically significantly decrease proliferation of a cell expressing ErbB3 relative to the proliferation in the absence of the antibody.
  • the proliferation of a cell expressing ErbB3 e.g ., a cancer cell
  • Cellular proliferation can be assayed using art recognized techniques which measure rate of cell division, the fraction of cells within a cell population undergoing cell division, and/or rate of cell loss from a cell population due to terminal differentiation or cell death (e.g., using a cell titer glow assay or thymidine incorporation).
  • the ability to decrease levels of ErbB3 on cell surfaces refers to the ability of an antibody or antigen binding portion thereof to statistically significantly reduce the amount of ErbB3 found on the surface of a cell which has been exposed to the antibody relative to an untreated (control) cell.
  • a decrease in levels of ErbB3 on cell surfaces may result from increased internalization of ErbB3 (or increased ErbB3 endocytosis).
  • the antibody or antigen binding portion thereof decreases cell surface expression of ErbB3 by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or 100% and/or increases internalization of the ErbB3 receptor by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or 100% relative to the cell surface expression or internalization in the absence of the antibody or antigen binding portion thereof (control).
  • the levels of ErbB3 on surfaces of cells and/or internalization of the ErbB3 receptor in the absence and the presence of an antibody or antigen-binding portion thereof can be readily measured using art recognized techniques, such as those described in Horst et al., supra and in the examples herein.
  • VEGF secretion of cells expressing ErbB3 refers to the ability of an antibody or an antigen-binding portion thereof to statistically significantly decrease VEGF secretion of a cell expressing ErbB3 relative to the VEGF secretion in the absence of the antibody.
  • the VEGF secretion of a cell expressing ErbB3 may be decreased by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or 100% when the cells are contacted with the antibody or antigen binding portion thereof of the present invention, relative to the VEGF secretion measured in the absence of the antibody or antigen binding portion thereof (control).
  • VEGF secretion can be assayed using art recognized techniques, such as those described herein.
  • the phrase "inhibition of the migration of cells expressing ErbB3,” as used herein, refers to the ability of an antibody or an antigen-binding portion thereof to statistically significantly decrease the migration of a cell expressing ErbB3 relative to the migration of the cell in the absence of the antibody.
  • the migration of a cell expressing ErbB3 (e.g ., a cancer cell) may be decreased by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or 100% when the cells are contacted with the antibody or antigen binding portion thereof of the present invention, relative to cell migration measured in the absence of the antibody or antigen binding portion thereof (control).
  • Cell migration can be assayed using art recognized techniques, such as those described herein.
  • inhibitortion of spheroid growth of cells expressing ErbB3 refers to the ability of an antibody or an antigen-binding portion thereof to statistically significantly decrease the migration of a cell expressing ErbB3 relative to the migration of the cell in the absence of the antibody.
  • the migration of a cell expressing ErbB3 may be decreased by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or 100% when the cells are contacted with the antibody or antigen binding portion thereof of the present invention, relative to cell migration measured in the absence of the antibody or antigen binding portion thereof (control).
  • Cell migration can be assayed using art recognized techniques, such as those described herein.
  • antibody or “immunoglobulin,” as used interchangeably herein, includes whole antibodies and any antigen binding fragment (i.e ., “antigen-binding portion") or single chains thereof.
  • An “antibody” comprises at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as V H ) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, CH1, CH2 and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as V L ) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • V H and V L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each V H and V L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g ., effector cells) and the first component (Clq) of the classical complement system.
  • Exemplary antibodies of the invention include antibodies#1, 3 and 14, and antigen-binding portions thereof.
  • antibody portion refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g ., ErbB3). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the V L , V H , CL and CH1 domains; (ii) a F(ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V H and CH1 domains; (iv) a Fv fragment consisting of the V L and V H domains of a single arm of an antibody, (v) a dAb including VH and VL domains; (vi) a dAb fragment ( Ward et al.
  • V H domain a dAb which consists of a VH or a VL domain
  • CDR an isolated complementarity determining region
  • ix a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker.
  • the two domains of the Fv fragment, V L and V H are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V L and V H regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g ., Bird et al.
  • scFv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody.
  • antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
  • Antigen-binding portions can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e ., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Monoclonal antibodies can be prepared using any art recognized technique and those described herein such as, for example, a hybridoma method, as described by Kohler et al.
  • recombinant antibody refers to antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g ., a mouse) that is transgenic or transchromosomal for immunoglobulin genes (e.g ., human immunoglobulin genes) or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the antibody, e.g ., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial antibody library ( e.g ., containing human antibody sequences) using phage display, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of immunoglobulin gene sequences (e.g ., human immunoglobulin genes) to other DNA sequences.
  • a host cell transformed to express the antibody e.g ., from a transfectoma
  • combinatorial antibody library e.g ., containing human
  • Such recombinant antibodies may have variable and constant regions derived from human germline immunoglobulin sequences.
  • such recombinant human antibodies can be subjected to in vitro mutagenesis and thus the amino acid sequences of the V H and V L regions of the recombinant antibodies are sequences that, while derived from and related to human germline V H and V L sequences, may not naturally exist within the human antibody germline repertoire in vivo .
  • chimeric immunoglobulin refers to an immunoglobulin or antibody whose variable regions derive from a first species and whose constant regions derive from a second species. Chimeric immunoglobulins or antibodies can be constructed, for example by genetic engineering, from immunoglobulin gene segments belonging to different species.
  • human antibody is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences as described, for example, by Kabat et al. (See Kabat, et al. (1991) Sequences of proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 ). Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences.
  • human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g ., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo ).
  • human antibody as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • the human antibody can have at least one ore more amino acids replaced with an amino acid residue, e.g ., an activity enhancing amino acid residue which is not encoded by the human germline immunoglobulin sequence.
  • the human antibody can have up to twenty positions replaced with amino acid residues which are not part of the human germline immunoglobulin sequence. In a particular embodiment, these replacements are within the CDR regions as described in detail below.
  • humanized immunoglobulin refers to an immunoglobulin or antibody that includes at least one humanized immunoglobulin or antibody chain ( i.e ., at least one humanized light or heavy chain).
  • humanized immunoglobulin chain or “humanized antibody chain” (i.e ., a “humanized immunoglobulin light chain” or “humanized immunoglobulin heavy chain”) refers to an immunoglobulin or antibody chain ( i.e ., a light or heavy chain, respectively) having a variable region that includes a variable framework region substantially from a human immunoglobulin or antibody and complementarity determining regions (CDRs) (e.g., at least one CDR, preferably two CDRs, more preferably three CDRs) substantially from a non-human immunoglobulin or antibody, and further includes constant regions (e.g ., at least one constant region or portion thereof, in the case of a light chain, and preferably three constant regions in the case of a
  • CDRs complementarity determining regions
  • humanized variable region refers to a variable region that includes a variable framework region substantially from a human immunoglobulin or antibody and complementarity determining regions (CDRs) substantially from a non-human immunoglobulin or antibody.
  • CDRs complementarity determining regions
  • bispecific or “bifunctional antibody” is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites.
  • Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. See, e.g ., Songsivilai & Lachmann, (1990) Clin. Exp. Immunol. 79, 315-321 ; Kostelny et al. (1992) J. Immunol. 148, 1547-1553 .
  • a bispecific antibody according to the present invention includes binding sites for both ErbB3 and IGF1-R (i.e ., insulin-like growth factor 1-receptor).
  • a bispecific antibody according to the present invention includes binding sites for both ErbB3 and C-MET.
  • a bispecific antibody includes a binding site for ErbB3 and a binding site for ErbB2, ERbB3, ErbB4, EGFR, Lewis Y, MUC-1, EpCAM, CA125, prostate specific membrane antigen, PDGFR- ⁇ , PDGFR- ⁇ , C-KIT, or any of the FGF receptors.
  • a heterologous antibody is defined in relation to the transgenic non-human organism or plant producing such an antibody.
  • an "isolated antibody,” as used herein, is intended to refer to an antibody which is substantially free of other antibodies having different antigenic specificities (e.g ., an isolated antibody that specifically binds to ErbB3 is substantially free of antibodies that specifically bind antigens other than ErbB3).
  • an isolated antibody is typically substantially free of other cellular material and/or chemicals.
  • a combination of "isolated" monoclonal antibodies having different ErbB3 binding specificities are combined in a well defined composition.
  • isotype refers to the antibody class (e.g ., IgM or IgG1) that is encoded by heavy chain constant region genes.
  • an antibody or antigen binding portion thereof is of an isotype selected from an IgG1, an IgG2, an IgG3, an IgG4, an IgM, an IgA1, an IgA2, an IgAsec, an IgD, or an IgE antibody isotype.
  • a monoclonal antibody of the invention is of the IgG1 isotype.
  • a monoclonal antibody of the invention is of the IgG2 isotype.
  • isotype switching refers to the phenomenon by which the class, or isotype, of an antibody changes from one Ig class to one of the other Ig classes.
  • nonswitched isotype refers to the isotypic class of heavy chain that is produced when no isotype switching has taken place; the CH gene encoding the nonswitched isotype is typically the first CH gene immediately downstream from the functionally rearranged VDJ gene. Isotype switching has been classified as classical or non-classical isotype switching.
  • Classical isotype switching occurs by recombination events which involve at least one switch sequence regions in a gene encoding an antibody.
  • Non-classical isotype switching may occur by, for example, homologous recombination between human ⁇ ⁇ and human ⁇ ⁇ ( ⁇ -associated deletion).
  • Alternative non-classical switching mechanisms such as intertransgene and/or interchromosomal recombination, among others, may occur and effectuate isotype switching.
  • switch sequence refers to those DNA sequences responsible for switch recombination.
  • a "switch donor” sequence typically a ⁇ switch region, will be 5' ( i.e., upstream) of the construct region to be deleted during the switch recombination.
  • the "switch acceptor” region will be between the construct region to be deleted and the replacement constant region (e.g ., ⁇ , ⁇ , etc.). As there is no specific site where recombination always occurs, the final gene sequence will typically not be predictable from the construct.
  • an “antigen” is an entity (e.g ., a proteinaceous entity or peptide) to which an antibody or antigen-binding portion thereof binds.
  • an antigen is ErbB3 or a ErbB3-like molecule.
  • an antigen is human ErbB3.
  • epitopes refers to a site on an antigen to which an immunoglobulin or antibody specifically binds.
  • Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.
  • An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation.
  • Methods of determining spatial conformation of epitopes include techniques in the art and those described herein, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g. , Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996 ).
  • antibodies that bind the same or an overlapping epitope as the antibodies of the present invention i.e ., antibodies that compete for binding to ErbB3, or bind epitopes which overlap with epitopes bound by the antibodies described herein, i.e., an epitope located on domain I of ErbB3.
  • Antibodies that recognize the same epitope can be identified using routine techniques such as an immunoassay, for example, by showing the ability of one antibody to block the binding of another antibody to a target antigen, i.e ., a competitive binding assay.
  • Competitive binding is determined in an assay in which the immunoglobulin under test inhibits specific binding of a reference antibody to a common antigen, such as ErbB3.
  • RIA solid phase direct or indirect radioimmunoassay
  • EIA solid phase direct or indirect enzyme immunoassay
  • sandwich competition assay see Stahli et al., (1983) Methods in Enzymology 9:242
  • solid phase direct biotin-avidin EIA see Kirkland et al., (1986) J. Immunol. 137:3614
  • solid phase direct labeled assay solid phase direct labeled sandwich assay
  • solid phase direct label RIA using I-125 label see Morel et al., (1988) Mol. Immunol.
  • the terms “specific binding,” “specifically binds,” “selective binding,” and “selectively binds,” mean that an antibody or antigen-binding portion thereof, exhibits appreciable affinity for a particular antigen or epitope and, generally, does not exhibit significant cross-reactivity with other antigens and epitopes.
  • “Appreciable” or preferred binding includes binding with an affinity of at least 10 6 , 10 7 , 10 8 , 10 9 M -1 , or 10 10 M -1 . Affinities greater than 10 7 M -1 , preferably greater than 10 8 M -1 are more preferred.
  • a preferred binding affinity can be indicated as a range of affinities, for example, 10 6 to 10 10 M -1 , preferably 10 7 to 10 10 M -1 , more preferably 10 8 to 10 10 M -1 .
  • An antibody that "does not exhibit significant cross-reactivity" is one that will not appreciably bind to an undesirable entity (e.g ., an undesirable proteinaceous entity).
  • an antibody or antigen-binding portion thereof that specifically binds to ErbB3 will appreciably bind that ErbB3 molecule but will not significantly react with other ErbB molecules and non-ErbB proteins or peptides.
  • Specific or selective binding can be determined according to any art-recognized means for determining such binding, including, for example, according to Scatchard analysis and/or competitive binding assays.
  • K D is intended to refer to the dissociation equilibrium constant of a particular antibody-antigen interaction or the affinity of an antibody for an antigen.
  • the antibody or antigen binding portion thereof according to the present invention binds an antigen (e.g ., ErbB3) with an affinity (K D ) of 50 nM or better (i.e., or less) ( e.g ., 40 nM or 30 nM or 20 nM or 10 nM or less), as measured using a surface plasmon resonance assay or a cell binding assay.
  • an antibody or antigen binding portion thereof binds ErbB3 with an affinity (K D ) of 8 nM or better (e.g ., 7 nM, 6 nM, 5 nM, 4 nM, 2 nM, 1.5 nM, 1.4 nM, 1.3 nM, 1nM or less), as measured by a surface plasmon resonance assay or a cell binding assay.
  • K D affinity
  • an antibody or antigen binding portion thereof binds an antigen (e.g ., ErbB3) with an affinity (K D ) of approximately less than 10 -7 M, such as approximately less than 10 -8 M, 10 -9 M or 10 -10 M or even lower when determined by surface plasmon resonance (SPR) technology in a BIACORE 3000 instrument using recombinant ErbB3 as the analyte and the antibody as the ligand, and binds to the predetermined antigen with an affinity that is at least two-fold greater than its affinity for binding to a non-specific antigen (e.g ., BSA, casein) other than the predetermined antigen or a closely-related antigen.
  • a non-specific antigen e.g ., BSA, casein
  • K off is intended to refer to the off rate constant for the dissociation of an antibody from the antibody/antigen complex.
  • EC50 refers to the concentration of an antibody or an antigen-binding portion thereof, which induces a response, either in an in vitro or an in vivo assay, which is 50% of the maximal response, i.e ., halfway between the maximal response and the baseline.
  • glycosylation pattern is defined as the pattern of carbohydrate units that are covalently attached to a protein, more specifically to an immunoglobulin protein.
  • naturally-occurring refers to the fact that an object can be found in nature.
  • a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring.
  • rearranged refers to a configuration of a heavy chain or light chain immunoglobulin locus wherein a V segment is positioned immediately adjacent to a D-J or J segment in a conformation encoding essentially a complete V H or V L domain, respectively.
  • a rearranged immunoglobulin gene locus can be identified by comparison to germline DNA; a rearranged locus will have at least one recombined heptamer/nonamer homology element.
  • V segment configuration refers to the configuration wherein the V segment is not recombined so as to be immediately adjacent to a D or J segment.
  • nucleic acid molecule is intended to include DNA molecules and RNA molecules.
  • a nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA.
  • isolated nucleic acid molecule as used herein in reference to nucleic acids encoding antibodies or antibody portions ( e.g ., V H , V L , CDR3) that bind to ErbB3, is intended to refer to a nucleic acid molecule in which the nucleotide sequences encoding the antibody or antibody portion are free of other nucleotide sequences encoding antibodies that bind antigens other than ErbB3, which other sequences may naturally flank the nucleic acid in human genomic DNA.
  • modifying is intended to refer to changing one or more amino acids in the antibodies or antigen-binding portions thereof.
  • the change can be produced by adding, substituting or deleting an amino acid at one or more positions.
  • the change can be produced using known techniques, such as PCR mutagenesis.
  • an antibody or an antigen-binding portion thereof identified using the methods of the invention can be modified, to thereby modify the binding affinity of the antibody or antigen-binding portion thereof to ErbB3.
  • the present invention also encompasses "conservative amino acid substitutions" in the sequences of the antibodies of the invention, i.e ., nucleotide and amino acid sequence modifications which do not abrogate the binding of the antibody encoded by the nucleotide sequence or containing the amino acid sequence, to the antigen, i.e ., ErbB3.
  • Conservative amino acid substitutions include the substitution of an amino acid in one class by an amino acid of the same class, where a class is defined by common physicochemical amino acid side chain properties and high substitution frequencies in homologous proteins found in nature, as determined, for example, by a standard Dayhoff frequency exchange matrix or BLOSUM matrix.
  • non-conservative amino acid substitution refers to the substitution of an amino acid in one class with an amino acid from another class; for example, substitution of an Ala, a class II residue, with a class III residue such as Asp, Asn, Glu, or Gln.
  • mutations can be introduced randomly along all or part of an anti-ErbB3 antibody coding sequence, such as by saturation mutagenesis, and the resulting modified anti-ErbB3 antibodies can be screened for binding activity.
  • a "consensus sequence” is a sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related sequences (See e.g ., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987 ). In a family of proteins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence.
  • a “consensus framework” of an immunoglobulin refers to a framework region in the consensus immunoglobulin sequence.
  • the consensus sequence for the CDRs of can be derived by optimal alignment of the CDR amino acid sequences of ErbB3 antibodies of the present invention.
  • nucleic acids For nucleic acids, the term "substantial homology" indicates that two nucleic acids, or designated sequences thereof, when optimally aligned and compared, are identical, with appropriate nucleotide insertions or deletions, in at least about 80% of the nucleotides, usually at least about 90% to 95%, and more preferably at least about 98% to 99.5% of the nucleotides. Alternatively, substantial homology exists when the segments will hybridize under selective hybridization conditions, to the complement of the strand.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.
  • the percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989 )) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol.
  • the nucleic acid and protein sequences of the present invention can further be used as a "query sequence" to perform a search against public databases to, for example, identify related sequences.
  • Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10 .
  • Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402 .
  • the default parameters of the respective programs e.g ., XBLAST and NBLAST.
  • the nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.
  • a nucleic acid is "isolated” or “rendered substantially pure” when purified away from other cellular components or other contaminants, e.g ., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well known in the art. See, F. Ausubel, et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987 ).
  • nucleic acid compositions of the present invention while often in a native sequence (except for modified restriction sites and the like), from either cDNA, genomic or mixtures thereof may be mutated, in accordance with standard techniques to provide gene sequences. For coding sequences, these mutations, may affect amino acid sequence as desired.
  • DNA sequences substantially homologous to or derived from native V, D, J, constant, switches and other such sequences described herein are contemplated (where "derived" indicates that a sequence is identical or modified from another sequence).
  • operably linked refers to a nucleic acid sequence placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • “operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase.
  • a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence.
  • operably linked means that the DNA sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.
  • operably linked indicates that the sequences are capable of effecting switch recombination
  • vector is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as "recombinant expression vectors"(or simply, "expression vectors”).
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector may be used interchangeably.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g ., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • recombinant host cell (or simply “host cell”), as used herein, is intended to refer to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.
  • treat refers to therapeutic or preventative measures described herein.
  • the methods of “treatment” employ administration to a subject, an antibody or antigen binding portion of the present invention, for example, a subject having a disease or disorder associated with ErbB3 dependent signaling or predisposed to having such a disease or disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disease or disorder or recurring disease or disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • disease associated with ErbB3 dependent signaling includes disease states and/or symptoms associated with a disease state, where increased levels of ErbB3 and/or activation of cellular cascades involving ErbB3 are found. It is understood that ErbB3 heterodimerizes with other ErbB proteins such as, EGFR and ErbB2, when increased levels of ErbB3 are found. Accordingly, the term “disease associated with ErbB3 dependent signaling,” also includes disease states and/or symptoms associated with disease states where increased levels of EGFR/ErbB3 and/or ErbB2/ErbB3 heterodimers are found.
  • disease associated with ErbB3 dependent signaling refers to any disorder, the onset, progression or the persistence of the symptoms of which requires the participation of ErbB3.
  • Exemplary ErbB3-mediated disorders include, but are not limited to, for example, cancer.
  • cancer and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
  • cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastric cancer, pancreatic cancer, glial cell tumors such as glioblastoma and neurofibromatosis, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, melanoma, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer.
  • a cancer treated or diagnosed using the methods of the present invention is selected from melanoma, breast cancer, ovarian cancer, renal carcinoma, gastrointestinal/colon cancer, lung cancer, and prostate cancer.
  • an effective amount refers to that amount of an antibody or an antigen binding portion thereof that binds ErbB3, which is sufficient to effect treatment, prognosis or diagnosis of a disease associated with ErbB3 dependent signaling, as described herein, when administered to a subject.
  • a therapeutically effective amount will vary depending upon the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.
  • the dosages for administration can range from, for example, about 1 ng to about 10,000 mg, about 5 ng to about 9,500 mg, about 10 ng to about 9,000 mg, about 20 ng to about 8,500 mg, about 30 ng to about 7,500 mg, about 40 ng to about 7,000 mg, about 50 ng to about 6,500 mg, about 100 ng to about 6,000 mg, about 200 ng to about 5,500 mg, about 300 ng to about 5,000 mg, about 400 ng to about 4,500 mg, about 500 ng to about 4,000 mg, about 1 ⁇ g to about 3,500 mg, about 5 ⁇ g to about 3,000 mg, about 10 ⁇ g to about 2,600 mg, about 20 ⁇ g to about 2,575 mg, about 30 ⁇ g to about 2,550 mg, about 40 ⁇ g to about 2,500 mg, about 50 ⁇ g to about 2,475 mg, about 100 ⁇ g to about 2,450 mg, about 200 ⁇ g to about 2,425 mg, about 300 ⁇ g to about 2,000, about 400 ⁇ g to about 1,175 mg
  • patient includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.
  • the term "subject” includes any human or non-human animal.
  • the methods and compositions of the present invention can be used to treat a subject having cancer.
  • the subject is a human.
  • non-human animal includes all vertebrates, e.g ., mammals and non-mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, reptiles, etc .
  • sample refers to tissue, body fluid, or a cell from a patient or a subject. Normally, the tissue or cell will be removed from the patient, but in vivo diagnosis is also contemplated.
  • a tissue sample can be taken from a surgically removed tumor and prepared for testing by conventional techniques.
  • lymphomas and leukemias lymphocytes, leukemic cells, or lymph tissues can be obtained and appropriately prepared.
  • Other patient samples including urine, tear drops, serum, cerebrospinal fluid, feces, sputum, cell extracts etc . can also be useful for particular tumors.
  • anti-cancer agent and “antineoplastic agent” refer to drugs used to treat malignancies, such as cancerous growths. Drug therapy may be used alone, or in combination with other treatments such as surgery or radiation therapy. Several classes of drugs may be used in cancer treatment, depending on the nature of the organ involved. For example, breast cancers are commonly stimulated by estrogens, and may be treated with drugs which inactive the sex hormones. Similarly, prostate cancer may be treated with drugs that inactivate androgens, the male sex hormone.
  • Anti-cancer agents of the present invention include, among others, the following agents: Anti-Cancer Agent Comments Examples Antibodies (a) antibodies other than anti-ErbB3 antibodies; and (b) anti-ErbB3 antibodies which bind different epitopes Antibodies which bind IGF-1R (insulin-like growth factor type 1 receptor), which is expressed on the cell surface of must human cancers A12 (fully humanized mAb) 19D12 (fully humanized mAb) CP751-871 (fully humanized mAb) H7C10 (humanized mAb) alphaIR3 (mouse) scFV/FC (mouse/human chimera) EM/164 (mouse) Antibodies which bind EGFR (epidermal growth factor receptor); Mutations affecting EGFR expression or activity could result in cancer Matuzumab (EMD72000) Erbitux® / Cetuximab (Imclone) Vectibix® / Panitumumab (Amgen)
  • Flourouracil (5-FU) Capecitabine / XELODA® (HLR Roche) 5-Trifluoromethyl-2'-deoxyuridine Methotrexate sodium (Trexall) (Barr) Raltitrexed / Tomudex® (AstraZaneca) Pemetrexed / Alimta® (Lilly) Tegafur Cytosine Arabinoside (Cytarabine, Ara-C) / Thioguanine® (GlaxoSmithKline) 5-azacytidine 6-mercaptopurine (Mercaptopurine, 6-MP) Azathioprine / Azasan® (AAIPHARMA LLC) 6-thioguanine (6-TG) / Purinethol® (TEVA) Pentostatin / Nipent® (Hospira Inc.) Fludarabine phosphate / Fludara® (Bayer Health Care) Cladribine (2-CdA, 2-chlorodeoxyadenosine)
  • Doxorubicin HCL/Doxil® (Alza) Daunorubicin citrate / Daunoxome® (Gilead) Mitoxantrone HCL/Novantrone (EMD Serono) Actinomycin D Etoposide / Vepesid® (BMS)/ Etopophos® (Hospira, Bedford, Teva Parenteral, Etc.) Topotecan HCL / Hycamtin® (GlaxoSmithKline) Teniposide (VM-26) / Vumon® (BMS) Irinotecan HCL(CPT-11) / Camptosar® (Pharmacia & Upjohn) Microtubule targeting agents Microtubules are one of the components of the cytoskeleton.
  • Microtubules serve as structural components within cells and are involved in many cellular processes including mitosis, cytokinesis, and vesicular transport.
  • Vincristine / Oncovin® Vinblastine sulfate/Velban®(discontinued) (Lilly) Vinorelbine tartrate / Navelbine® (PierreFabre) Vindesine sulphate / Eldisine® (Lilly) Paclitaxel / Taxol® (BMS) Docetaxel / Taxotere® (Sanofi Aventis US) Nanoparticle paclitaxel (ABI-007) / Abraxane® (Abraxis BioScience, Inc.) Ixabepilone / IXEMPRATM (BMS) Kinase inhibitors Tyrosine kinases are enzymes within the cell that function to attach phosphate groups to the amino acid tyrosine.
  • these compounds provide a tool for controlling cancerous Imatinib mesylate/Gleevec(Novartis) Sunitinib malate / Sutent® (Pfizer) Sorafenib tosylate / Nexavar® (Bayer) Nilotinib hydrochloride monohydrate / Tasigna® (Novartis) cell growth.
  • Protein synthesis inhibitors Induces cell apoptosis L-asparaginase / Elspar® (Merck & Co.) Immunotherapeutic agents induces cancer patients to exhibit immune responsiveness Alpha interferon Angiogenesis Inhibitor / Avastin® (Genentech) IL-2 ⁇ Interleukin 2 (Aldesleukin) / Proleukin ® (Chiron) IL-12 ⁇ Interleukin 12 Hormones Hormone therapies associated with associated with aging menopause and seek to increase the amount of certain hormones in your body to compensate for age- or disease-related hormonal declines. Hormone therapy as a cancer treatment either reduces the level of specific hormones or alters the cancer's ability to use these hormones to grow and spread.
  • Predinsolone Dexamethasone / Decadron® (Wyeth) Aromatose inhibitors Includes imidazoles Ketoconazole mTOR inhibitors The mTOR signaling The mTOR signaling pathway was originally discovered during studies of the immunosuppressive agent rapamycin.
  • Chemotherapeutic agents Adriamycin, 5-Fluorouracil, Cytoxin, Bleomycin, Mitomycin C, Daunomycin, Carminomycin, Aminopterin, Dactinomycin, Mitomycins, Esperamicins
  • One or more anti-cancer agents may be administered either simultaneously or before or after administration of an antibody or antigen binding portion thereof of the present invention.
  • Monoclonal antibodies of the invention can be produced using a variety of known techniques, such as the standard somatic cell hybridization technique described by Kohler and Milstein (1975) Nature 256: 495 , viral or oncogenic transformation of B lymphocytes or phage display technique using libraries of human antibody genes.
  • the antibodies are fully human monoclonal antibodies.
  • a hybridoma method is used for producing an antibody that binds ErbB3.
  • a mouse or other appropriate host animal can be immunized with a suitable antigen in order to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the antigen used for immunization.
  • lymphocytes may be immunized in vitro. Lymphocytes can then be fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell ( Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986 )).
  • Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods ( Goding, Monoclonal Antibodies:Principles and Practice, pp. 59-103 (Academic Press, 1986 )). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal.
  • the monoclonal antibodies secreted by the subclones can be separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • antibodies and antibody portions that bind ErbB3 can be isolated from antibody phage libraries generated using the techniques described in, for example, McCafferty et al., Nature, 348:552-554 (1990 ). Clackson et al., Nature, 352:624-628 (1991 ), Marks et al., J. Mol. Biol., 222:581-597 (1991 ) and Hoet et al (2005) Nature Biotechnology 23, 344-348 ; U.S. Patent Nos. 5,223,409 ; 5,403,484 ; and 5,571,698 to Ladner et al. ; U.S. Patent Nos.
  • the monoclonal antibody or antigen binding portion thereof that binds ErbB3 is produced using the phage display technique described by Hoet et al. , supra.
  • This technique involves the generation of a human Fab library having a unique combination of immunoglobulin sequences isolated from human donors and having synthetic diversity in the heavy-chain CDRs is generated. The library is then screened for Fabs that bind to ErbB3.
  • human monoclonal antibodies directed against ErbB3 can be generated using transgenic or transchromosomic mice carrying parts of the human immune system rather than the mouse system (see e . g ., Lonberg, et al. (1994) Nature 368(6474): 856-859 ; Lonberg, N. et al. (1994), supra ; reviewed in Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101 ; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. 13: 65-93 , and Harding, F. and Lonberg, N. (1995) Ann. N.Y. Acad. Sci. 764:536-546 .
  • human antibodies of the invention can be raised using a mouse that carries human immunoglobulin sequences on transgenes and transchomosomes, such as a mouse that carries a human heavy chain transgene and a human light chain transchromosome (see e.g., PCT Publication WO 02/43478 to Ishida et al. ).
  • transgenic animal systems expressing human immunoglobulin genes are available in the art and can be used to raise anti-ErbB3 antibodies of the invention.
  • an alternative transgenic system referred to as the Xenomouse (Abgenix, Inc.) can be used; such mice are described in, for example, U.S. Patent Nos. 5,939,598 ; 6,075,181 ; 6,114,598 ; 6, 150,584 and 6,162,963 to Kucherlapati et al.
  • mice carrying both a human heavy chain transchromosome and a human light chain tranchromosome can be used; as described in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727 .
  • cows carrying human heavy and light chain transchromosomes have been described in the art ( Kuroiwa et al. (2002) Nature Biotechnology 20:889-894 ) and can be used to raise anti-ErbB3 antibodies of the invention.
  • antibodies of the present invention can be prepared using a transgenic plant and/or cultured plant cells (such as, for example, tobacco, maize and duckweed) that produce such antibodies.
  • transgenic tobacco leaves expressing antibodies or antigen binding portions thereof can be used to produce such antibodies by, for example, using an inducible promoter (see, e . g ., Cramer et al., Curr. Top. Microbol. Immunol. 240:95 118 (1999 )).
  • transgenic maize can be used to express such antibodies and antigen binding portions thereof (see, e.g., Hood et al., Adv. Exp. Med. Biol. 464:127 147 (1999 )).
  • Antibodies can also be produced in large amounts from transgenic plant seeds including antibody portions, such as single chain antibodies (scFv's), for example, using tobacco seeds and potato tubers (see, e.g., Conrad et al., Plant Mol. Biol. 38:101 109 (1998 )).
  • Methods of producing antibodies or antigen binding portions in plants can also be found in, e . g ., Fischer et al., Biotechnol. Appl. Biochem. 30:99 108 (1999 ), Ma et al., Trends Biotechnol. 13:522 7 (1995 ); Ma et al., Plant Physiol. 109:341 6 (1995 ); Whitelam et al., Biochem. Soc. Trans. 22:940 944 (1994 ) and U.S. Patent Nos. 6,040,498 and 6,815,184 .
  • the binding specificity of monoclonal antibodies or portions thereof that bind ErbB3 prepared using any technique including those disclosed here, can be determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunoabsorbent assay
  • the binding affinity of a monoclonal antibody or portion thereof also can be determined by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980 ).
  • an ErbB3 antibody or portion thereof produced using any of the methods discussed above may be further altered or optimized to achieve a desired binding specificity and/or affinity using art recognized techniques, such as those described herein.
  • partial antibody sequences derived from an ErbB3 antibody may be used to produce structurally and functionally related antibodies.
  • antibodies interact with target antigens predominantly through amino acid residues that are located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside of CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that include CDR sequences from the specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e . g ., Riechmann, L.
  • Such framework sequences can be obtained from public DNA databases that include germline antibody gene sequences.
  • an anti-ErbB3 antibody of the invention can be used to create structurally related anti-ErbB3 antibodies that retain at least one functional property of the antibodies of the invention, e.g., inhibiting EGF-like ligand mediated phosphorylation of ErbB3; inhibiting one or more of heregulin, epiregulin, epigen or biregulin-mediated signaling through ErbB3; inhibiting proliferation or cells expressing ErbB3; and/or decreasing levels of ErbB3 on cell surfaces.
  • one or more CDR regions selected from SEQ ID NOs:7-12, SEQ IDNOs:13-18, SEQ ID NOs:19-24, SEQ ID NOs:39-44, and SEQ ID NOs:45-50 is combined recombinantly with known human framework regions and CDRs to create additional, recombinantly-engineered, anti-ErbB3 antibodies of the invention.
  • the heavy and light chain variable framework regions can be derived from the same or different antibody sequences.
  • antibodies are generated that include the heavy and/or light chain CDR3s of the particular antibodies described herein (e.g., SEQ ID NOs:9, 15, 21, 41, 47 and/or SEQ ID NOs:12, 18, 24, 44, 50).
  • the antibodies can further include the heavy and/or light chain CDR1 and/or CDR2s of the antibodies of the present invention ( e .
  • the CDRs, 2, and/or 3 regions of the engineered antibodies described above can comprise the exact amino acid sequence(s) as those disclosed herein (e . g ., CDRs of Ab #6, Ab #3, Ab #14, Ab #17, or Ab #19, set forth in SEQ ID NOs:7-12, 13-18, 19-24, 39-44, and 45-50, respectively).
  • the engineered antibody may be composed of one or more CDRs that are, for example, 90%, 95%, 98%, 99% or 99.5% identical to one or more CDRs of Ab #6, Ab #3 or Ab #14.
  • one or more residues of a CDR may be altered to modify binding to achieve a more favored on-rate of binding.
  • an antibody having ultra high binding affinity of, for example, 10 10 M -1 or more can be achieved.
  • Affinity maturation techniques well known in the art and those described herein, can be used to alter the CDR region(s) followed by screening of the resultant binding molecules for the desired change in binding. Accordingly, as CDR(s) are altered, changes in binding affinity as well as immunogenicity can be monitored and scored such that an antibody optimized for the best combined binding and low immunogenicity are achieved.
  • modifications can also be made within one or more of the framework regions, FR1, FR2, FR3 and FR4, of the heavy and/or the light chain variable regions of an antibody, so long as these modifications do not eliminate the binding affinity of the antibody.
  • the antibody is further modified with respect to effector function, so as to enhance the effectiveness of the antibody in treating cancer, for example.
  • cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region.
  • the homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med. 176:1191-1195 (1992 ) and Shopes, B. J. Immunol. 148:2918-2922 (1992 ).
  • Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al.
  • an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al. Anti-Cancer Drug Design 3:219-230 (1989 ).
  • bispecific antibodies and immunoconjugates are also encompassed by the present invention.
  • Bispecific antibodies of the present invention include at least one binding specificity for ErbB3 and at least one binding specificity for another antigen, such as the product of an oncogene.
  • Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab') 2 bispecific antibodies).
  • bispecific antibodies are well known in the art (see, e.g., WO 05117973 and WO 06091209 ).
  • production of full length bispecific antibodies can be based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (see, e . g ., Millstein et al., Nature, 305:537-539 (1983 )).
  • Further details of generating bispecific antibodies can be found, for example, in Suresh et al., Methods in Enzymology, 121:210 (1986 ) and in Brennan et al., Science, 229: 81 (1985 ), which describes a chemical linkage process for making bispecific antibodies.
  • bispecific antibodies have been produced using leucine zippers (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992 )).
  • Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported (see, e . g ., Gruber et al., J. Immunol., 152:5368 (1994 )).
  • the bispecific antibody comprises a first antibody or binding portion thereof which binds to ErbB3 and a second antibody or binding portion thereof which binds to ErbB2, ERbB3, ErbB4, EGFR, IGF1-R, C-MET, Lewis Y, MUC-1, EpCAM, CA125, prostate specific membrane antigen, PDGFR- ⁇ , PDGFR- ⁇ , C-KIT, or any of the FGF receptors.
  • Immunoconjugates of the present invention can be formed by conjugating the antibodies or antigen binding portions thereof described herein to another therapeutic agent.
  • Suitable agents include, for example, a cytotoxic agent (e.g., a chemotherapeutic agent), a toxin (e . g . an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), and/or a radioactive isotope (i.e. , a radioconjugate).
  • a cytotoxic agent e.g., a chemotherapeutic agent
  • a toxin e.g . an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof
  • a radioactive isotope i.e. , a radioconjugate
  • Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes.
  • a variety of radionuclides are available for the production of radioconjugated anti-ErbB3 antibodies. Examples include 212 Bi, 131 I, 131 In, 90 Y and 186 Re.
  • Immunoconjugates of the invention can be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).
  • SPDP N-succinimidyl-3-(2-pyri
  • a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987 ).
  • Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody (see, e . g ., WO94/11026 ).
  • antibodies or antigen binding portions that bind ErbB3 can be screened for various properties, such as those described herein, using a variety of assays that are well known in the art.
  • the antibodies or antigen binding portions thereof are screened for the ability to inhibit EGF-like ligand mediated phosphorylation of ErbB3.
  • This can be done by treating cells expressing ErbB3 with an EGF-like ligand in the presence and absence of the antibody or antigen binding portion thereof. The cells can then be lysed and the crude lysates can be centrifuged to remove insoluble material. ErbB3 phosphorylation can be measured, for example, by Western blotting followed by probing with an anti-phosphotyrosine antibody as described in Kim et al. , supra and the Examples below.
  • the antibodies and antigen binding portions are further screened for one or more of the following properties: (1) inhibition of ErbB3-ligand (e . g ., heregulin, epiregulin, epigen or biregulin) mediated signaling through ErbB3; (2) inhibition of proliferation of cells expressing ErbB3; (3) the ability to decrease levels of ErbB3 on cell surface ( e .
  • ErbB3-ligand e . g ., heregulin, epiregulin, epigen or biregulin
  • Inhibition of one or more of heregulin, epiregulin, epigen or biregulin-mediated signaling through ErbB3 can be readily measured using routine assays, such as, described in Horst et al. supra.
  • the ability of an antibody or antigen binding portion thereof to inhibit heregulin, epiregulin, epigen or biregulin-mediated signaling through ErbB3 can be measured by kinase assays for known substrates of ErbB3 such as, for example, SHC and PI3K, as described in, for example, Horst et al. supra , Sudo et al., (2000) Methods Enzymol, 322:388-92 ; and Morgan et al. (1990) Eur. J.
  • Biochem., 191:761-767 following stimulation by one or more of heregulin, epiregulin, epigen or biregulin.
  • cells expressing ErbB3 can be stimulated with one or more of heregulin, epiregulin, epigen or biregulin, and incubated with a candidate antibody or antigen-binding portion thereof.
  • Cell lysates subsequently prepared from such cells can be immunoprecipitated with an antibody for a substrate of ErbB3 (or a protein in a cellular pathway involving ErbB3) such as, for example, an anti-JNK-1 antibody, and assayed for kinase activity ( e .
  • a decrease in or complete disappearance in level or activity (e . g ., kinase activity) of a ErbB3 substrate or protein in a pathway involving ErbB3 in the presence of the antibody or antigen binding portion thereof, relative to the level or activity in the absence of the antibody or antigen binding portion thereof, is indicative of an antibody or antigen binding portion which inhibits one or more of heregulin, epiregulin, epigen or biregulin-mediated signaling.
  • the antibody or antigen binding portion thereof inhibits ErbB3-ligand (e . g ., heregulin, epiregulin, epigen or biregulin) mediated signaling by decreasing the binding of one or more of heregulin, epiregulin, epigen or biregulin to ERbB3.
  • ErbB3-ligand e . g ., heregulin, epiregulin, epigen or biregulin
  • cells which express ErbB3 e . g . MALME-3M cells, as described in the Examples infra
  • a labeled ErbB3-ligand e . g ., radiolabeled heregulin, epiregulin, epigen or biregulin
  • the antibody or antigen binding portion thereof inhibits heregulin, epiregulin, epigen or biregulin binding to ErbB3, then a statistically significantly decrease in the amount of label recovered (e . g ., radiolabeled heregulin, epiregulin, epigen or biregulin), relative to the amount in the absence of the antibody or antigen binding portion thereof, will be observed.
  • label recovered e . g ., radiolabeled heregulin, epiregulin, epigen or biregulin
  • the antibody or antigen binding portion thereof may inhibit the binding of the ErbB3-ligand (e . g ., heregulin, epiregulin, epigen or biregulin) by any mechanism.
  • the antibody or antigen binding portion thereof may inhibit binding of the ErbB3 ligand ( e . g ., one or more of heregulin, epiregulin, epigen or biregulin) to ErbB3 by binding to the same site or an overlapping site on ErbB3 as the ErbB3 ligand.
  • the antibody or antigen binding portion thereof may inhibit binding of an ErbB3 ligand by altering or distorting the conformation of ErbB3, such that it is unable to bind to the ErbB3 ligand.
  • Antibodies and antigen binding portions thereof that decrease levels of ErbB3 on cell surfaces can be identified by their ability to downregulate ErbB3 on tumor cells.
  • the antibodies or antigen binding portions thereof decrease ErbB3 cell surface expression by inducing internalization (or increasing endocytosis) of Erbb3.
  • ErbB3 can be biotinylated and the number of ErbB3 molecules on the cell surface can be readily determined, for example, by measuring the amount of biotin on a monolayer of cells in culture in the presence or absence of an antibody or antigen binding portion thereof, for example, as described in, e.g. , Waterman et al., J. Biol. Chem.
  • Antibodies or antigen binding portions thereof of the present invention can also be tested for their ability to inhibit proliferation of cells expressing ErbB3, for example, tumor cells, using art recognized techniques, such as the Cell Titer Glow Assay described in the Examples below (also see, e . g ., Macallan et al., Proc. Natl. Acad Sci. (1998) 20;95(2):708-13 ; Perez et al. (1995) Cancer Research 55, 392-398 ).
  • the antibodies or antigen binding portions thereof are screened for the ability to inhibit VEGF secretion of cells expressing ErbB3. This can be done by using well-known assays, such as the VEGF ELISA kit available from R&D Systems (Minneapolis, MN, Cat.#DY293B). Similarly, the antibodies or portions can be screened for the ability to inhibit the migration of cells expressing ErbB3 ( e . g ., MCF-7 cells) using a trans-well assay (Millipore Corp., Billerica, MA, Cat # ECM552) as described herein.
  • a trans-well assay Millipore Corp., Billerica, MA, Cat # ECM552
  • the antibodies or antigen binding portions thereof are screened for the ability to inhibit spheroid growth of cells expressing ErbB3. This can be done by using an assay which approximates conditions of a developing tumor growth (see, e.g., Herman et al. (2007) Journal of Biomolecular Screening Electronic publication ) as described herein.
  • Antibodies or antigen binding portions thereof that bind to the same or overlapping epitopes as one or more antibodies of the present invention can also be identified using standard techniques known in the art and described herein. For example, in order to screen for antibodies which bind to the same or an overlapping epitope on ErbB3 bound by an antibody of interest, a cross-blocking assay, such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988 ), can be performed.
  • the present invention provides a composition, e . g ., a pharmaceutical composition, containing one or a combination of monoclonal antibodies, or antigen-binding portion(s) thereof, of the present invention, formulated together with a pharmaceutically acceptable carrier.
  • the compositions include a combination of multiple ( e . g ., two or more) isolated antibodies of the invention, which bind different epitopes on ErbB3.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).
  • the active compound i.e., antibody, bispecific and multispecific molecule, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.
  • a “pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S.M., et al. (1977) J. Pharm. Sci. 66:1-19 ). Examples of such salts include acid addition salts and base addition salts.
  • Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenylsubstituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like.
  • nontoxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like
  • nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenylsubstituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like.
  • Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.
  • compositions of the invention can be administered alone or in combination therapy, i.e., combined with other agents.
  • the combination therapy can include a composition of the present invention with at least one or more additional therapeutic agents, such as the anti-cancer agents described infra.
  • additional therapeutic agents such as the anti-cancer agents described infra.
  • the pharmaceutical compositions of the invention can also be administered in conjunction with radiation therapy and/or surgery.
  • a composition of the present invention can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results.
  • the active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978 .
  • the compound may be administered to a subject in an appropriate carrier, for example, liposomes, or a diluent.
  • suitable diluents include saline and aqueous buffer solutions.
  • Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes ( Strejan et al. (1984) J. Neuroimmunol. 7:27 ).
  • Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • the use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • compositions typically must be sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freezedrying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Dosage regimens are adjusted to provide the optimum desired response (e . g ., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
  • the human antibodies of the invention may be administered once or twice weekly by subcutaneous injection or once or twice monthly by subcutaneous injection.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
  • antioxidants examples include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), le
  • formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods known in the art of pharmacy.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 0.001 per cent to about ninety percent of active ingredient, preferably from about 0.005 per cent to about 70 per cent, most preferably from about 0.01 per cent to about 30 per cent.
  • Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.
  • Dosage forms for the topical or transdermal administration of compositions of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • adjuvants which are well-known in the art include, for example, inorganic adjuvants (such as aluminum salts, e.g., aluminum phosphate and aluminumhydroxide), organic adjuvants (e.g., squalene), oil-based adjuvants, virosomes (e.g., virosomes which contain a membrane-bound heagglutinin and neuraminidase derived from the influenza virus).
  • inorganic adjuvants such as aluminum salts, e.g., aluminum phosphate and aluminumhydroxide
  • organic adjuvants e.g., squalene
  • oil-based adjuvants e.g., virosomes which contain a membrane-
  • Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
  • the compounds of the present invention are administered as pharmaceuticals, to humans and animals, they can be given alone or as a pharmaceutical composition containing, for example, 0.001 to 90% (more preferably, 0.005 to 70%, such as 0.01 to 30%) of active ingredient in combination with a pharmaceutically acceptable carrier.
  • the compounds of the present invention which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • a suitable daily dose of a composition of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect.
  • Such an effective dose will generally depend upon the factors described above. It is preferred that administration be intravenous, intramuscular, intraperitoneal, or subcutaneous, preferably administered proximal to the site of the target.
  • the effective daily dose of a therapeutic composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition).
  • compositions can be administered with medical devices known in the art.
  • a therapeutic composition of the invention can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. Patent Nos. 5,399,163 , 5,383,851 , 5,312,335 , 5,064,413 , 4,941,880 , 4,790,824 , or 4,596,556 .
  • a needleless hypodermic injection device such as the devices disclosed in U.S. Patent Nos. 5,399,163 , 5,383,851 , 5,312,335 , 5,064,413 , 4,941,880 , 4,790,824 , or 4,596,556 .
  • Examples of well-known implants and modules useful in the present invention include: U. S. Patent No. 4,487,603 , which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No.
  • the monoclonal antibodies of the invention can be formulated to ensure proper distribution in vivo.
  • the blood-brain barrier (BBB) excludes many highly hydrophilic compounds.
  • the therapeutic compounds of the invention cross the BBB (if desired)
  • they can be formulated, for example, in liposomes.
  • liposomes For methods of manufacturing liposomes, see, e . g ., U.S. Patents 4,522,811 ; 5,374,548 ; and 5,399,331 .
  • the liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., V.V. Ranade (1989) J. Clin. Pharmacol. 29:685 ).
  • Exemplary targeting moieties include folate or biotin (see, e . g ., U.S. Patent 5,416,016 to Low et al. ); mannosides ( Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038 ); antibodies ( P.G. Bloeman et al. (1995) FEBS Lett. 357:140 ; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180 ); surfactant protein A receptor ( Briscoe et al. (1995) Am. J. Physiol.
  • the present invention also provides methods of using antibodies and antigenbinding portions thereof that bind ErbB3 in a variety of ex vivo and in vivo diagnostic and therapeutic applications.
  • antibodies of the invention can be used for treating a disease associated with ErbB3 dependent signaling, including a variety of cancers.
  • the present invention provides a method for treating a disease associated with ErbB3 dependent signaling by administering to a subject an antibody or antigen binding portion thereof of the invention in an amount effective to treat the disease.
  • Suitable diseases include, for example, a variety of cancers including, but not limited to, melanoma, breast cancer, ovarian cancer, renal carcinoma, gastrointestinal cancer, colon cancer, lung cancer, and prostate cancer.
  • the antibody can be administered alone or with another therapeutic agent which acts in conjunction with or synergistically with the antibody to treat the disease associated with ErbB3 mediated signaling.
  • therapeutic agents include, for example, the anticancer agents described infra (e.g., cytotoxins, chemotherapeutic agents, small molecules and radiation).
  • the present invention provides a method for diagnosing a disease (e . g ., a cancer) associated with ErbB3 upregulation in a subject, by contacting antibodies or antigen binding portions of the invention (e.g., ex vivo or in vivo) with cells from the subject, and measuring the level of binding to ErbB3 on the cells. Abnormally high levels of binding to ErbB3 indicate that the subject has a disease associated with ErbB3 upregulation.
  • a disease e . g ., a cancer
  • antibodies or antigen binding portions of the invention e.g., ex vivo or in vivo
  • kits comprising antibodies and antigen binding portions thereof of the invention which optionally include instructions for use in treating or diagnosing a disease associated with ErbB3 upregulation and/or ErbB3 dependent signaling.
  • the kits may include a label indicating the intended use of the contents of the kit.
  • label includes any writing, marketing materials or recorded material supplied on or with the kit, or which otherwise accompanies the kit.
  • the practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, recombinant DNA technology, immunology (especially, e . g ., antibody technology), and standard techniques in polypeptide preparation.
  • conventional techniques of chemistry, molecular biology, recombinant DNA technology, immunology (especially, e . g ., antibody technology), and standard techniques in polypeptide preparation See, e . g ., Sambrook, Fritsch and Maniatis, Molecular Cloning: Cold Spring Harbor Laboratory Press (1989 ); Antibody Engineering Protocols (Methods in Molecular Biology), 510, Paul, S., Humana Pr (1996 ); Antibody Engineering: A Practical Approach (Practical Approach Series, 169), McCafferty, Ed., Irl Pr (1996 ); Antibodies: A Laboratory Manual, Harlow et al., C. S.H.L. Press, Pub.
  • a cryopulverizer (Covaris Inc) was used for the pulverization of tumors. Tumors were stored in special bags (pre-weighed before the addition of the tumor) and placed in liquid nitrogen while handling them. For small tumors, 200 uL of Lysis buffer was first added to the bag containing the tumor, frozen in liquid nitrogen and then pulverized to improve the recovery of the tumor from the bag. Pulverized tumors were transferred to 2 mL Eppendorf tubes and placed in liquid nitrogen until ready for further processing
  • Tumors were lysed in Lysis buffer supplemented with protease and phosphatase inhibitors. Lysis Buffer was added to the tumor aliquots in a final concentration of about 62.5 mg/mL. Tumor samples were homogenized by vortexing for 30 sec and incubating on ice for about 30 min. The lysates were spun for about 10 min in Qiagen Qiashredder columns for further homogenization of the samples. Cleared lysates were aliquoted into fresh tubes for further processing.
  • ELISA reagents for the total and phospho-ErbB3 ELISAs were purchased from R&D Systems as Duoset kits.
  • 96-well Nunc Maxisorb plates were coated with 50 uL of an antibody and incubated overnight at room temperature. Next morning, plates were washed 3 times with 1000 ⁇ l/well in the BioTek plate washer with PBST (0.05% Tween-20). Plates were subsequently blocked for about an 1 hr at room temperature with 2 % BSA in PBS. The plates were washed 3 times with 1000 ⁇ l/well in the BioTek plate washer with PBST (0.05%Tween-20). 50 ⁇ L of cell lysates and standards diluted in 50% Lysis buffer and 1%BSA were used in duplicates for further processing.
  • Binding of the various Fabs to MALME-3M cells was next examined using about 500 nM of the Fabs and a 1:750 dilution of a goat anti-human Alexa 647 secondary antibody. As shown in Figures 1A and 1B , several candidate Fabs exhibited appreciable staining of MALME-3M cells.
  • VH and VL regions were reformatted using expression constructs for expression as an IgG1 or IgG2 isotype.
  • the constructs included a Selexis backbone which has a cassette designed for substitution of the appropriate heavy and light chain sequences.
  • the Selexis vectors included a CMV promoter and a matching poly-A signal.
  • nucleic acid sequences for the codon-optimized VH and VL of Ab #6 are set forth in SEQ ID NOs:25 and 26, respectively, and those for Ab #3 are set forth in SEQ ID NOs:27 and 28, respectively, as shown in Figure 22 .
  • the dissociation constants of the anti-ErbB3 antibodies were measured using two independent techniques, i . e ., a Surface Plasmon Resonance Assay and a cell binding assay using MALME-3M cells.
  • the Surface Plasmon Resonance Assay (also referred to as the Flexchip assay) was performed as described in Wassaf et al. (2006) Analytical Biochem., 351:241-253 .
  • the cell binding assay for determining the K D values of Ab #6 and Ab #3 was performed as follows.
  • MALME-3M cells were detached with 2 mls trypsin-EDTA + 2 mls RMPI + 5mM EDTA at room temperature for 5 minutes.
  • Complete RPMI (10 mls) was added immediately to the trypsinized cells, resuspended gently and spun down in a Beckman tabletop centrifuge at 1100 rpm for 5 minutes.
  • Cells were resuspended in BD stain buffer (PBS + 2% fetal bovine serum + 0.1% sodium azide, Becton Dickinson) at a concentration of 2 x 10 6 cells per ml and 50 ⁇ l (1 x 10 5 cells) aliquots were plated in a 96-well titer plate.
  • a 150 ⁇ l solution of 200 nM anti-ErbB3 antibody (Ab #6 or Ab #3) in BD stain buffer was prepared in an eppendorf tube and serially diluted 2-fold into 75 ⁇ l BD stain buffer. The concentrations of the diluted antibody ranged from 200 nM to 0.4 nM. 50 ⁇ l aliquots of the different protein dilutions were then added directly to the 50 ul cell suspension giving the final concentrations of 100 nM, 50 nM, 25 nM, 12 nM, 6 nM, 3 nM, 1.5 nM, 0.8 nM, 0.4 nM and 0.2 nM of the antibody.
  • MFI values and the corresponding concentrations of the anti-ErbB3-antibodies were plotted on the y-axis and x-axis, respectively.
  • the K D of the molecule was determined by GraphPad Prism using the one-site binding model for a non-linear regression curve.
  • the binding specificity of an IgG2 isotype of Ab #6 to ErbB3 was assayed using ELISA as follows. Identification of the epitope bound by Ab #6 was also analyzed.
  • 96-well Nunc Maxisorb plates were coated with 50 ⁇ l of 5 ⁇ g/ml protein (recombinant human ErbB3, recombinant human EGFR or unrelated protein (BSA)) and incubated overnight at room temperature. The next morning, plates were washed 3 times with 1000 ⁇ l/well of PBST (0.05% Tween-20) in the BioTek plate washer. The wells were blocked for 1 hr at room temperature with 2 % BSA in PBS. The plates were washed 3 times with 1000 ⁇ l/well of PBST (0.05%Tween-20) in the BioTek plate washer.
  • PBST recombinant human ErbB3, recombinant human EGFR or unrelated protein
  • the plates were washed plate 3 times with 1000 ⁇ l/well of PBST (0.05%Tween-20) in the BioTek plate washer. 50 ⁇ L of Supersignal Pico ELISA substrate was added and the plate was read on the Fusion plate reader. The data was analyzed using the EXCEL program. Duplicate samples were averaged and the error bars represent the standard deviation between the two replicates.
  • a fragment (truncation mutant) corresponding to amino acid residues 20-202 of ErbB3 was cloned into the yeast display vector pYD2 (a modified version of pYD1 (Invitrogen) with a stop codon engineered in front of the His tag) between the Nhe and BsiWI restriction sites.
  • the plasmid was transformed into the yeast strain EBY100 (Invitrogen) and clones containing the plasmid selected on Trp- selective medium. The clone was grown in glucose containing medium overnight at 30°C and expression of the ErbB3 truncation mutant was induced by transfer to a galactose-containing medium for 2 days at 18°C.
  • Yeast displaying the ErbB3 truncation mutant were stained with 50 nM of Ab #6, followed by a goat anti-human antibody labeled with Alexa dye-647. A separate sample was stained with the goat anti-human antibody only to show that there is no non-specific binding to yeast of the secondary antibody. Analysis was performed by flow cytometry on the FACS Calibur cell sorter (BD Biosciences).
  • Ab #6 bound to the truncation mutant i.e., amino acid residues 20-202 of ErbB3.
  • MALME-3M cells were seeded in 96 well tissue culture plates and grown in RPMI-1640 media supplemented with antibiotics, 2mM L-glutamine and 10% fetal bovine serum (FBS) for 24 hours at 37°C and 5% carbon dioxide. Media was then switched to RPMI-1640 MEDIA with antibiotics, 2mM L-glutamine with and without the antibody at concentrations of 1 ⁇ M, 250nM, 63nM, 16nM, 4.0nM, 1.0nM, 240pM, 61pM and 15pM.
  • FBS fetal bovine serum
  • Cells were grown for 24 hours at 37°C and 5% carbon dioxide, washed with cold PBS, then harvested with mammalian protein extract (MPER) lysis (Pierce, 78505) buffer containing 150mM NaCl, 5mM sodium pyrophosphate, 10uM bpV (phen), 50uM phenalarsine, 1mM sodium orthovanadate, and protease inhibitor cocktail (Sigma, P714). Cell lysates were diluted two-fold with 4% bovine serum albumin in phosphate buffered saline with 0.1% tween-20, then analyzed by ELISA with mouse anti-human ErbB3 capture antibody and biotinylated mouse anti-human ErbB3 secondary detection antibody. Signal was generated with streptavidin conjugated to horseradish-peroxidase reacted with chemiluminescent substrate (Pierce, 37070). ELISAS were visualized using a luminometer.
  • MPER mammalian protein extract
  • Ab #6 decreased total ErbB3 levels by about 46.9% in MALME-3M cells in vitro, as measured by ELISA. Media containing no serum and antibody was used as control.
  • MALME-3M cells were trypsinized from a 15 cm dish and washed once with RPMI + 10% fetal bovine serum. Cell pellets were resuspended at a density of 1 x 10 6 cells per ml. Two aliquots of 2 x 10 5 cells were added to a 12-well tissue culture plate and resuspended in a final volume of 800 ul RPMI + 10% fetal bovine serum. To one well, Ab #6 IgG1 or Ab #6IgG2 isotype was added to a final concentration of 100 nM (treated sample) and to the other well, an equivalent volume of PBS was (untreated sample) was added.
  • treated and untreated cells were trypsinized, washed and incubated with 100 nM of Ab #6 in BD stain buffer for 30 minutes on ice.
  • Cells were washed twice with 1 ml BD stain buffer and incubated with 100 ul of a 1:500 dilution of Alexa 647-labeled goat anti-human Alexa 647 for 45 minutes on ice.
  • Cells were then washed and resuspended in 300 ul BD stain buffer + 0.5 ug/ml propidium iodide. Analysis of 10,000 cells was done in a FACScalibur flow cytometer using the FL4 channel.
  • both IgG1 and IgG2 isotypes of Ab #6 downregulated ErbB3 on MALME-3M cells by about 62% and about 66%, respectively.
  • MALME-3M cells were trypsinized from a 15 cm dish and washed once with RPMI + 10% fetal bovine serum. Cell pellets were resuspended at a density of 1 x 10 6 cells per ml. Two aliquots of 2 x 10 5 cells were added to a 12-well tissue culture plate and resuspended in a final volume of 800 ⁇ l RPMI + 10% fetal bovine serum.
  • the anti-ErbB3 antibody was added to a final concentration of 100 nM (treated sample) and to the other well, an equivalent volume of PBS was (untreated sample) was added. The following day, treated and untreated cells were trypsinized, washed and incubated with 100 nM anti-ErbB3 antibody in BD stain buffer for 30 minutes on ice. Cells were washed twice with 1 ml BD stain buffer and incubated with 100 ⁇ l of a 1:500 dilution of Alexa 647-labeled goat anti-human Alexa 647 for 45 minutes on ice.
  • T-cell deficient nu/nu mice (3-4 week old female mice originated at NIH; outbred; albino background) were purchased from Charles River Labs (Wilmington, MA).
  • MALME-3M cells for implantation were grown in culture (RPMI media, 10% FBS, L-glutamine and antibiotics, 37°C, 5% CO2) to about 80% confluency before harvesting. Cells were kept on ice until implantation. Mice were implanted via subcutaneous injection with 100ul MALME-3M cells on the right flank and allowed to recover while being monitored for initial tumor growth.
  • mice were dosed with IgG2a (Sigma, M7769-5MG) by intravenous injection. Mice were dosed intra-peritoneally every other day with either 15 ⁇ g or 100 ⁇ g of antibody number 6 and tumors were measured three times per week and recorded in a Microsoft EXCEL spreadsheet.
  • Ab #6 caused downregulation of total ErbB3 as soon as 24 hours post-injection in tumors treated with either IgG1 or IgG2 isotype of Ab #6).
  • PBS was used as a control
  • the samples were pulverized in a cryopulverizer (Covaris Inc). Tumors were stored in special bags (pre-weighed before the addition of the tumor) and placed in liquid nitrogen while handling them. For small tumors, 200 ⁇ L of Lysis buffer was first added to the bag with the tumor, frozen in liquid nitrogen and then pulverized to improve the recovery of the tumor from the bag. Pulverized tumors were transferred to 2 ml Eppendorf tubes and placed in liquid nitrogen until lysed. Tumors were lysed in Lysis buffer supplemented with protease and phosphotase inhibitors. Lysis Buffer was added to the tumor aliquots in a final concentration of 62.5 mg/ml.
  • Tumor samples were homogenized by vortexing for 30 seconds and letting them sit on ice for 30 min. The lysates were spun for 10 minutes in Qiagen Qiashredder columns for further homogenization of the samples. Cleared lysates were aliquoted into fresh tubes.
  • the BCA assay was performed as set forth in the materials and methods section supra.
  • the total levels of ErbB3 were determined by ELISA.
  • the ELISA reagents were purchased from R&D Systems as Duoset kits.
  • 96-well Nunc Maxisorb plates were coated with 50 ⁇ l of respective capture antibody and incubated overnight at room temperature. The next morning, the plates were washed 3 times with 1000 ⁇ l/well in a BioTek plate washer with PBST (0.05% Tween-20) and then blocked for 1 hour at room temperature with 2 % BSA in PBS. The plates were then washed three times with 1000 ⁇ l/well in the BioTek plate washer with PBST (0.05%Tween-20). Lysates (50 ⁇ l) and standards were diluted in 50% Lysis buffer and 1%BSA; all samples were run in duplicate.
  • MALME3M, ACHN and NCI/ADRr cells were seeded in 96 well tissue culture plates and grown in RPMI-1640 media supplemented with antibiotics, 2mM L-glutamine and 10% fetal bovine serum (FBS) for 24 hours at 37 degrees Celsius and 5% carbon dioxide. Media was then switched to RPMI-1640 media with antibiotics, 2mM L-glutamine and with and without the antibody at 1uM, 250nM, 63nM, 16nM, 4.0nM, 1.0nM, 240pM, 61pM and 15pM concentrations.
  • FBS fetal bovine serum
  • Cells were grown for 96 hours at 37°C and 5% carbon dioxide, then harvested with CellTiter-Glo® Luminescent Cell Viability Assay (Promega, G7573) and analyzed on a luminometer. Media containing no serum and antibody was used as control.
  • Ab #6 inhibited proliferation of MALME-3M cells ( Figure 9 ), ADRr ovarian cancer cells ( Figure 10 ) and ACHN cells ( Figure 11 ) which express ErbB3.
  • Ab # 6 inhibited proliferation of MALME-3M cells by about 19.6%, as measured using the Cell Titer Glow assay, and inhibited proliferation of ADRr ovarian cancer cells by about 30.5%.
  • Ab # 6 inhibited proliferation of ACHN cells by about 25.4%.
  • Example 5 supra The samples were pulverized using the technique described in Example 5 supra , with respect to Figure 8 .
  • the BCA assay was performed as set forth in the Materials and Methods section supra , and the ELISA assay was performed as described in Example 5 supra with respect to Figure 8 .
  • Ovarian ADRr cells were preincubated with Ab #6 for 30 minutes prior to stimulation with 50 mM BTC, 10 mM HRG or 333 nM TGF- ⁇ . Following pre incubation, the media was removed and the cells were stimulated for 5 minutes at 37°C, 5% CO2 with 50nM BTC or 333nM TGF- ⁇ (for PE498). HRG controls (5 minutes, 5nM), 10% serum and 0% serum controls were also used.
  • OVCAR 5 and OVCAR 8 cell lines were obtained from the National. Cancer Institute, Division of Cancer Treatment and Diagnostics ("DCTD").
  • DCTD Cancer Treatment and Diagnostics
  • the ELISA was performed as described in the Materials and Methods section supra .
  • ADRr cells or MALME-3M cells (1 x 10 5 ) were pre-incubated with 25 ⁇ M of anti-ErbB3 Ab # 6 or 25 ⁇ M of Erbitux (as control) in 50 ⁇ l BD stain buffer for 30 minutes on ice. After 30 minutes, 50 ⁇ l of 400 nM biotinylated BTC was added to the cells and incubated for another 30 minutes on ice. This gave a final concentration of 12.5 ⁇ M antibodies and 200 nM BTC.
  • MALME-3M cells were seeded in 96 well tissue culture plates and grown in RPMI-1640 media supplemented with antibiotics, 2mM L-glutamine and 10% fetal bovine serum (FBS) for 24 hours at 37°C and 5% carbon dioxide. Cells were serum starved in RPMI-1640 media with antibiotics and 2mM L-glutamine for 24 hours at 37°C and 5% carbon dioxide.
  • FBS fetal bovine serum
  • Cells were pre-treated with and without the anti-ErbB3 antibody (IgG2 isotype of Ab #6) at 1 ⁇ M, 250nM, 63nM, 16nM, 4.0nM, 1.0nM, 240pM and 61pM concentrations for 30 minutes then stimulated with HRG1-betal-ECD for 10 minutes at 37°C and 5% carbon dioxide.
  • IgG2 isotype of Ab #6 the anti-ErbB3 antibody
  • MPER mammalian protein extract
  • lysis Pierce, 78505
  • buffer containing 150mM NaCl 5mM sodium pyrophosphate, 10uM bpV (phen), 50 ⁇ M phenalarsine, 1mM sodium orthovanadate, and protease inhibitor cocktail (Sigma, P714).
  • Cell lysates were diluted two-fold with 4% bovine serum albumin in phosphate buffered saline with 0.1% tween-20, then analyzed by ELISA for AKT (a downstream effector of ErbB3) and ErbB3 phosphorylation.
  • AKT a downstream effector of ErbB3
  • ErbB3 phosphorylation a downstream effector of ErbB3 phosphorylation.
  • lysates were run on an ELISA plate with a capture antibody specific for AKT and biotinylated detection antibody specific to the phosphorylation site on serine 473 of AKT. Signal was generated with streptavidin conjugated to horseradish-peroxidase reacted with chemiluminescent substrate (Pierce, 37070).
  • chemiluminescent substrate Pierce, 37070
  • lysates were run on an ELISA plate with a capture antibody specific for ErbB3 and an anti-phosphotyrosine detection antibody conjugated to horseradish-peroxidase. This was then reacted with chemiluminescent substrate (Pierce, 37070). ELISAs were visualized using a luminometer.
  • Ab #6 was a potent inhibitor of heregulin-mediated signaling in MALME-3M cells, as measured by decreased phosphorylation of ErbB3 ( Figure 16A ) and AKT ( Figure 16B ). Notably, Ab #6 inhibited the phosphorylation of AKT by almost 100%.
  • mice T-cell deficient nu/nu mice (3-4 week old female mice originated at NIH; outbred; albino background) were purchased from Charles River Labs (Wilmington, MA) for xenograft studies.
  • ADRr cells for implantation were grown in culture (RPMI media, 10% FBS, L-glutamine and antibiotics, 37°C, 5% CO2) to about 85% confluency before harvesting. Cells were kept on ice until implantation. Mice were implanted via subcutaneous injection with 100 ⁇ l ADRr cells on the right flank and allowed to recover while being monitored for initial tumor growth.
  • mice were dosed with IgG2a (Sigma, M7769-5MG) by intravenous injection. Mice were dosed intra-peritoneally every third day with either 30 ⁇ g or 300 ⁇ g of Ab #6 and tumors were measured three times per week and recorded in a Microsoft Excel spreadsheet.
  • FIGs 17A-C The data from three different models derived from human tumor cell lines, ADRr (ovarian), Du145 (prostate) and OvCAR8 (ovarian) are shown in Figures 17A-C and Colo357 xenograft study is shown in Figure 17D .
  • the data from these studies demonstrated that a 300ug dose of Ab #6 every three days (Q3d) results in significant inhibition of tumor growth (p ⁇ 0.05 for multiple time points during the studies). Moreover, this inhibitory effect of Ab #6 was further elevated when the dose was increased to 600ug, Q3d, in the Du145 prostate cancer model as well as a renal and a pancreatic carcinoma xenograft model (ACHN and COL0357).
  • ADRr cells (1 x 10 5 ) were incubated with 10 ⁇ M of an anti-ErbB3 antibody (e.g., Ab #6 or Ab/Fab # 3) in 50 ⁇ l BD stain buffer for 30 minutes on ice. After 30 minutes, 50 ⁇ l of 40 nM biotinylated heregulin EGF was added to the cells and incubated for another 10 minutes on ice. This gave a final concentration of 5 ⁇ M antibody and 20 nM heregulin EGF.
  • an anti-ErbB3 antibody e.g., Ab #6 or Ab/Fab # 3
  • PE phycoerythrin
  • PE phycoerythrin
  • HB-EGF binds to ErbB on ADRr cells and Ab #6 does not inhibit this binding, evidencing that Ab #6 is specific for inhibiting the binding of ErbB3 ligands ( e . g ., heregulin and epiregulin) to ErbB3.
  • ErbB3 ligands e . g ., heregulin and epiregulin
  • Ab #6 shows a potent inhibitory effect at high levels in all three cell lines with the highest being in COLO-357 ( Fig. 24A ).
  • Ab #6 also shows a similar effect in vivo by inhibiting VEGF secretion in three different xenografts, the highest being in COLO-357 xenograft ( Fig. 24B ).
  • Inhibition of VEGF correlates with inhibition of ErbB3 phosphorylation ( Fig. 24 C) .
  • Inhibition of VEGF secretion also correlates with inhibition of angiogenesis of the tumor cells.
  • myeloma cell-secreted factors such as VEGF and bFGF, trigger angiogenesis (see, e.g., Leung et al. (1989) Science 246(4935):1306-9 ; Yen et al. (2000) Oncogene 19(31):3460-9 ).
  • Ab #6 inhibits spheroid growth in AdrR cells ( Fig. 26A ).
  • 3.4nM HRG stimulates spheroid growth and Ab #6 inhibits the HRG effect ( Fig. 26B ).
  • Spheroids derived from DU145 did not increase in size during 13 days of the experiment; however, growth was significantly stimulated by HRG1-beta 1.
  • 8uM Ab #6 inhibits HRG induced spheroid growth ( Fig. 26C ).
  • Ab #6 The ability of Ab #6 to inhibit the signaling induced by different ligands was examined. For example, the effect of Ab #6 on HRG and BTC binding to AdrR cells expressing ErbB3 receptor was tested. As shown in Figures 27A and B , using FACS - analysis, Ab #6 competes with HRG and not BTC for binding to AdrR cells. Accordingly, blocking by Ab #6 of HRG binding to ErbB3 would prevent signaling induced by HRG.
  • HGF Hepatocyte Growth Factor
  • ErbB3 phosphorylation in AdrR cells in a dose dependent manner as shown in Figure 28 .
  • Ab #6 inhibits HGF induced erbB3 phosphorylation.
  • AdrR cells were pre-incubated with buffer (control), or 250 nM Ab #6 for 60 minutes at room temperature, then treated with 10 nM HRG or 10 nM BTC or control buffer for 10 minutes.
  • the cells were lysed in 25 mM Tris, pH+7.5, 150 mM NaCl, 1mM EDTA, 1.0% Triton X-100, 1.0% CHAPS, 10% v/v glycerol, containing 0.2mM PMSF, 50mTU/mL aprotinin, and 100 uM leupeptin, and the crude lysate were centrifuged briefly to remove insoluble material.
  • the immunoprecipitates were then washed with ice-cold lysis buffer 3 times, resuspended in 30 ul of 2X SDS sample buffer, heat denatured at 95°C for 7 minutes and run on 4-12% Bis-Tris Gels. SDS-PAGE and electro-transferred to PVDF membrane in Tri-Glycine buffer with 10% MeOH. The membrane was blocked for 1 hour in 10ml of blocking buffer (Li-Cor Biosciences, Lincoln, NE, Cat# 927-40000) and then incubated with the anti-ErbB2 antibody at 1:1000 (Cell Signaling Technology, Danvers, MA, Cat # 29D8) in 10 ml of blocking buffer (Li-Cor Biosciences, Cat# 927-40000). The signal was detected using goat anti-rabbit IRDye800 at 1:5000 (2ul) in 10ml of blocking buffer (Li-Cor Biosciences, Cat# 927-40000).

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